Combinatorial Synthesis of Libraries of Macrocyclic Compounds Useful in Drug Discovery

ABSTRACT

A library of macrocyclic compounds of the formula (I) 
     
       
         
         
             
             
         
       
         
         
           
             where part (A) is a 
           
         
       
    
     
       
         
         
             
             
         
       
     
     bivalent radical, a —(CH 2 ) y — bivalent radical or a covalent bond;
         where part (B) is a       

     
       
         
         
             
             
         
       
     
     bivalent radical, a —(CH 2 ) z — bivalent radical, or a covalent bond;
         where part (C) is a       

     
       
         
         
             
             
         
       
     
     bivalent radical, a —(CH2) t — bivalent radical, or a covalent bond; and
         where part (T) is a —Y-L-Z— radical wherein Y is CH 2  or CO, Z is NH or O and L is a bivalent radical. These compounds are useful for carrying out screening assays or as intermediates for the synthesis of other compounds of pharmaceutical interest. A process for the preparation of these compounds in a combinatorial manner, is also disclosed.

RELATED APPLICATION DATA

The present application is a continuation application of U.S. patentapplication Ser. No. 13/218,784, filed Aug. 26, 2011, now allowed, whichis a continuation application of U.S. patent application Ser. No.11/615,332, filed Dec. 22, 2006, now U.S. Pat. No. 8,008,440, which is acontinuation application of U.S. patent application Ser. No. 09/679,331,filed Oct. 4, 2000, now U.S. Pat. No. 7,169,899, which claims priorityto Canadian Application No. 2,284,459, filed Oct. 4, 1999. Thedisclosures of each of which are incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The present invention relates to new macrocyclic compounds ofbiologically interesting structures. The invention also relates to aprocess for preparing these new compounds by lactam or Mitsunobucyclization.

BACKGROUND OF THE INVENTION

As everybody knows, medicinal chemistry research has been dramaticallytransformed by biotechnology. Previously synthetic chemistry and naturalproducts screening dominated drug research, but now molecular biologyhas become a driving force behind screening and the establishment ofmolecular targets.

Over the last years, most of the research in biotech companies has beendirected to peptide and protein therapeutics in spite of problemsassociated with their low bioavailability, rapid metabolism, and lack oforal activity. Because of these limitations, research groups continue torely upon chemical synthesis of nonpeptide substances for drugdiscovery, recognizing that small molecules are likely to remain themost viable avenue for the identification and optimization of potentialdrugs.

As is also known, combinatorial chemistry is a technique by which largechemical libraries can be generated by connecting together appropriatechemical building blocks in a systematic way. The ability to generatesuch large, chemically diverse libraries, either in a combinatiorialfashion or by any other high throughput parallel synthetic methods,combined with high throughput screening techniques, provides animmensely powerful tool for drug lead discovery and optimization.

Drug companies are increasingly interested in harnessing the ability ofcombinatorial synthesis to produce large collections (or libraries) ofmolecules to augment their existing sources of molecular diversity, andto fully exploit their capacity to capture millions of biological assaydata points annually using high throughput robotic screeninginstrumentation.

This new science is still in its infancy, and to date most successfulscaffold are derived from small heterocycles which are usuallysynthesized in very few steps. Thus several focused libraries have beenbuilt around bioactive cores such as benzodiazepines. However, thisapproach cannot be considered as a true method to generate innovativelead structures. Rather, it is a mean to optimize existing leads and isusually applied in drug development schemes.

Random libraries destined to search for innovative leads are very fewtoday. As one example, a library based on diketopiperazine yielded a newsubmicromolar lead for a neurokinin-2 receptor after screening of thislibrary on a variety of targets.

It is obvious that not all scaffolds may lead to potent drug candidates.Some very simple molecules requiring only one or two chemical steps mayseem very attractive due to the huge size of the libraries that can begenerated from them. Nonetheless, too simple molecules do not usuallyprovide useful leads since they tend to lack target specificity, aprerequisite for a molecule to become a drug.

A class of organic structures with outstanding pharmaceutical activityhas been termed as “macrocycle family”. Compounds like Taxol,Epothilone, Erythromycin, Neocarzinostatin, Rifampin and Amphotericinare either under clinical study or already marketed drugs and belong tothis important family. Most of these products are of natural origin,since they are not usually tackled by medicinal chemists due to lack ofknowledge associated with their synthesis.

Over the last years, the present inventors have developed expertise inthe field of macrocycles synthesis. With such an expertise, they havedeveloped a method of synthesis and evaluation of libraries of partiallypeptidic macrocycles which mimic β-turns, thereby making it possible toquickly explore huge quantities of conformationally restrictedstructures.

OBJECTS AND SUMMARY OF THE INVENTION

A first object of the present invention is to provide a process forpreparing macrocyclic compounds, which process can be carried out with alarge variety of functional groups and in the presence of a largevariety of solvent systems and resins, and thus can be used forpreparing a large variety of macrocyclic compounds of biologicallyinteresting structures that could be used for carrying out screeningassays or as instrumental for the synthesis of other macrocycliccompounds. Libraries of such synthetic compounds should actually be asattractive as the libraries of extracted natural products which arepresently used in high throughput biological screening assays.

Another object of the invention is to provide libraries of macrocycliccompounds incorporating two to five building units: one to fouramino-acids and a tether chain for controlling the overall shape of themolecule.

More specifically, the macrocyclic compounds of the invention have thegeneral formula (I):

-   -   where part (A) is a

bivalent radical having its —NH— group linked to the carbonyl group ofpart

a —(CH₂)_(y)— bivalent radical, or a covalent bond;

-   -   where part (B) is a

bivalent radical having its —NH— group linked to part (A), a —(CH₂)_(z)—bivalent radical, or a covalent bond;

-   -   where part (C) is a

bivalent radical having its —NH— group linked to part (B), a —(CH₂)_(t)—bivalent radical, or a covalent bond;

-   -   where part (T) is a —Y-L-Z— radical; and    -   where X is a monovalent group selected from the group consisting        of: —SO₂—Ar, —SO₂—CH₃, —SO₂—CF₃, —H, —COH, —CO—CH3, —CO—Ar,        —CO—R, —CO—NHR, —CO—NHAr, —CO—O-tBu, —CO—O—CH₂—Ar

-   -   Ar being an aromatic group or substituted aromatic group,    -   a being an integer selected from the group consisting of 0, 1        and 2,    -   R being a monovalent group —(CH₂)_(n)—CH₃ or —(CH₂)_(n)—Ar with        n being an integer from 1 to 16,    -   R₀, R₁, R₂, R₃ and R₄ being independently selected from the        group consisting of:

R₅, R₆₁ and R₆₂ each being a monovalent radical independently selectedfrom the group consisting of: —H, —SO₂—CH₃, —SO₂—CF₃, —COH, —COCH₃,—CO—Ar, —CO—R or —CO—NHR wherein R is defined as above, —CONHAr,—COO-tBu and —COO—CH2-Ar, said radical being or not substituted by atleast one monovalent group selected in the group consisting of:

-   -   —O—CH₃, —CH₃, —NO₂, —NH₂, —NH—CH₃, —N(CH₃)₂, —CO—OH, —CO—O—CH₃,        —CO—CH₃, —CO—NH₂, OH, F, Cl, Br and I;

R₇ being a monovalent radical selected from the group consisting of:

-   -   —H, —COH, —CO—CH₃, —CO—R wherein R is defined as above, —CO—Ar        and —CO-tBu, said radical being substituted or not by at least        one substituent selected from the group consisting of:        -   —O—CH₃, —CH₃, —NO₂, —NH₂, —NH—CH₃, —N(CH₃)₂, —CO—OH,            —CO—O—CH₃, —CO—CH₃, —CO—NH₂, OH, F, Cl, Br and I;

R₈ being a monovalent radical selected from the group consisting of:—OH, —NH₂, —OCH₃, —NHCH₃, —O-tBu and —O—CH₂—Ar, said radical substitutedor not by at least one group selected in the group consisting of:

-   -   —O—CH₃, —CH₃, —NO₂, —NH—CH₃, —N(CH₃)₂, —CO—OH, —CO—O—CH₃,        —CO—CH₃, —CO—NH₂, OH, F, Cl, Br, and I;

R₉ being a monovalent radical selected in the group consisting of: —H,-tBu, —CO—CH₃, —COAr, —CO—R wherein R is defined as above and —COH, saidradicals substituted or not by at least one monovalent group selectedfrom the group consisting of:

-   -   —O—CH₃, —CH₃, —NO₂, —NH—CH₃, —N(CH₃)₂, —CO—OH, —CO—O—CH₃,        —CO—CH₃, —CO—NH₂, OH, F, Cl, Br and, I;

where Y is a bivalent group —CH₂— or —CO—;

where Z is a bivalent group —NH— or —O—;

wherein x, y, z and t are integers each independently selected from thegroup consisting of 0, 1 and 2;

wherein L is a bivalent radical selected from the group consisting of:

-   -   —(CH₂)_(d)-A-(CH₂)_(j)—B—(CH₂)_(e)—, d and e being independently        an integer from 1 to 5, j being an integer from 0 to 5,        -   with A and B being independently selected from the group            consisting of:            -   —O—, —NH—, —NR— wherein R is defined as above, —S—,                —CO—, —SO—, —CO—O—, —O—CO—, —CO—NH—, —NH—CO—, —SO₂—NH—,                —NH—SO₂—,

-   -   —CH═CH— with the configuration Z or E,

-   -   with the substituent -G₂- in a 1,2, 1,3 or 1,4 position,    -   G₁ being selected from the group consisting of:        -   —O—, —NH—, —NR— wherein R is defined as above, —S—, —CH═CH—            with a Z configuration, and —CH═N—; and    -   G₂ being selected from the group consisting of:        -   —O—, —NH—, —CO—, —NR— wherein R is defined as above, —CO—O—,            —O—CO—, —CO—NH—, —NH—CO—, —SO₂—NH— and —NH—SO₂—.

Salts of said compounds are also within the scope of the invention.

As may be understood, the new macrocyclic compounds according to theinvention incorporate two to five building units, one to fouramino-acids units and a tether chain which controls the shape of themolecule.

These compounds display enhanced stability towards peptidases andexhibit facilitated cell penetration as compared to the correspondingopen chain linear equivalents.

Some of the compounds according to the invention include a β-turn motifwithin their rings:

It is known that β-turn is one of the three major motifs of peptide andprotein secondary structure. β-turn plays a key role in many biologicalmolecular recognition events including interactions between antigens andantibodies, peptide hormones and their receptors, and regulatory enzymesand their corresponding substrates. In order to attain high affinity andselective binding to a targeted receptor, a β-turn mimetic mustreproduce both the functionality and the orientation of the side chainsof the receptor-bound peptide ligand.

The inherent diversity in β-turn structure compounded with difficultiesin identifying the key residues responsible for binding, make the designof β-turn mimetics quite challenging.

As may be appreciated, the present invention permits to circumvent theaforementioned difficulties by providing compounds which, thanks totheir structure which incorporate numerous side chain combinations aswell as multiple different side chain orientations, can be used asβ-turn mimetics and evaluated accordingly.

Obviously, the preparation of libraries of β-turn mimetics represents agoal of the present invention. However, the latter is not exclusivelyrestricted to such compounds. There are numerous other compoundsaccording to the invention which include other interesting di- ortri-peptide motif with their structure whose active conformation need beprobed by our conformation restrictive approach.

As is known many adhesive proteins present in extracellular matrices andin the blood contain the tripeptide arginine-glycine-aspartic acid(RTGD) as their cell recognition site. These proteins includefibronectin, vitronectin, osteopontin, callagens, thrombospondin,fibrinogen and von Willebrand factor. The RGD sequence of each of theadhesive proteins is recognized by at least one member of a family ofstructurally related receptors, integrins, which are heterodimericproteins with two membrane-spanning sub-units. Some of these receptorsbind to the RGD sequence of a single adhesion protein only, whereasothers recognize groups of them. The conformation of the RGD sequence inthe individual proteins may be critical to this recognition specificity.On the cytoplasmic side of the plasma membrane the receptors connect theextracellular matrix to the cytoskeleton.

More than ten proved or suspected RGD-containing adhesion-promotingproteins have already been identified, and the integrin family includesat least as many receptors recognizing these proteins. Together theadhesion proteins and their receptors constitute a versatile recognitionsystem providing cells with anchorage, traction for migration, andsignals for polarity, position, differentiation and possibly growth.Compounds according to the invention containing the sequence Arg-Gly-Aspin a controlled topology could inhibit cell to cell adhesion processes.Such compounds could be important in the areas of antithrombotic andcancer research.

Also included within the scope of the invention are compounds of theabove mentioned formula (I) containing a biaryl bridge.

As can be appreciated, the compounds according to the invention havemuch flexibility and can adopt structures very different fromconventional β-turns, according to the nature of their spacer parts.This means that the scope of the invention is broad and molecularmodeling design allows the design of β and non-β-turns.

A main advantage of the invention is that the compounds of the formula(I) are neither too tight like small rings nor too loose like aliphaticchains.

The process according to the invention for preparing the above mentionedcompounds basically comprises the following steps:

-   -   a) preparing by coupling a first building block deriving from        natural or synthetic amino-acids, said first building block        being of the formula:

-   -   wherein X, R₁, A, B, C are defined as hereinabove,        -   Sp is

-   -   and P is —CH₃ or —CH₂-Ph where the coupling is carried out in        liquid phase and Sp is

-   -   and P is polystyrene when the coupling is carried out in solid        phase;    -   b) coupling the first building block prepared in step a) with a        second building block hereafter called “tether”, of the formula:

H—Y-L-Z-PGz

-   -   wherein Y, L and Z are defined as and PGz is a protective group;        and    -   c) removing the protection groups PGz from the compound obtained        in step b); and    -   d) carrying out a macrocyclization of the unprotected product        obtained in step c) and a cleavage if the preparing and coupling        steps (a), (b) were carried out in the solid phase in order to        obtain the requested compound of the formula (I).

As can be noted, this process uses lactam or Mitsunob cyclization toprepare the libraries of compounds according to the invention. It isvery versatile and can be carried out in a combinatorial manner, eitherin solid phase or in solution phase.

The invention and its advantages will be better understood upon readingthe following non restrictive detailed description and examples madewith reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table detailing the structure that may have the compounds ofthe formula (I) according to the invention.

FIGS. 2 a and 2 b are schematic illustrations of the sequence of stepsthat must be carried out to obtain the library of compounds of formula(8), which is part of the “family 2” libraries shown in FIG. 1 for whichY is a methylene group (—CH₂—), and the amino-acids at positions 1, 2and 3 are α-amino-acids (x, y, z=0).

FIG. 3 is a schematic illustration of the steps that must be carried outto obtain other libraries of compounds of formulae (9) to (12), whichare also parts of the “family 2” libraries shown in FIG. 1, for which Yis a methylene group (—CH₂—), and the amino-acids at positions 1, 2 and3 are α-amino-acids (x, y, z=0).

FIGS. 4 a and 4 b are schematic illustrations of the sequence of stepsthat must be carried out to obtain libraries of compounds of theformulae (17) and (18) which are parts of the “family 2” libraries shownin FIG. 1, for which Y is a carbonyl group (CO), and the amino-acids atpositions 1, 2 and 3 are α-amino-acids (x, y, z=0).

DETAILED DESCRIPTION OF THE INVENTION

As aforesaid, the process according to the invention is versatile enoughto prepare a large number macrocyclic compounds, the main “families” ofwhich are illustrated in FIG. 1. This process comprises the followingbasic steps:

-   -   a) preparing by coupling a first building block deriving from        natural or synthetic amino-acids,    -   b) coupling the first building block prepared in step a) with a        second building block called “tether”,    -   c) removing the protective group from the compound obtained in        step b), and    -   d) carrying out a macrocyclization of the unprotected product        obtained in step c) to obtain the requested compound.

As aforesaid, the process permits to prepare a large number of compoundseither in a solution phase or on a solid support using an IRORIcombinatorial chemistry set up or another set up like an Argonautapparatus.

In the synthesis shown in FIG. 2 a, a first suitably protectedamino-acid identified as “A” is activated as a thioester (solution phaseor solid phase) or as an oxime ester (Kaiser resin solid phase support)to give a compound of formula (1). The amine protection (PGα in the caseof α-amino-acids PGβ in the case of n-amino-acids and PGγ in the case ofγ-amino-acids) is removed to give the compound of formula (2). A secondamino-acid identified as “B” is then added in the same way to give acompound of formula (3), followed again by removal of the amineprotection to give a compound of formula (4). A third acid of formula(C) (FIG. 2 b) is coupled to the amine of formula (4) to yield asulfonamide of the formula (5), which is immediately coupled with analcohol of the formula (D) under Mitsunobu conditions to give a compoundof formula (6). This compound of formula (6) can also be obtaineddirectly from the compound of formula (4) by peptide coupling with anacid of formula (E). The terminal alcohol (Z═O) or amine (Z ═NH)protecting group (PGz) is then cleaved to give the corresponding alcoholor amine of formula (7), which can undergo cyclization and cleavage allat once to give requested compound according to the invention of formula(8).

As shown in FIG. 3, the orthogonal protections (PG1, PG2, PG3 and PG4when a fourth amino-acid is introduced) of the compound of formula (8)can be removed to yield the compound according to the invention offormula (9). Another compound according to the invention of formula (10)can be obtained from the compound of formula (8) by cleaving thesulfonamide portion of the molecules. The resulting free amine can becoupled with various acids (see X groups in FIG. 1) to yield a compoundof formula (11) according to the invention. Subsequent cleavage of theorthogonal protecting groups (PG0 when X is an amino-acid, PG1, PG2, PG3and PG4 when a fourth amino-acid is introduced) yields a compound offormula (12) according to the invention.

As shown in FIG. 4, it is also possible from the amine of formula (4) tocouple an amino-acid of formula (C′) to yield a compound of formula (13)according to the invention, whose amine protecting group (PGα, PGβ orPGγ) can be cleaved to give the amine of formula (14). A hydroxy-acid(Z═O) or amino-acid (Z═NH) of formula (D′) is then coupled to yield acompound according to the invention of formula (15). The terminalalcohol (Z=0) or amine (Z═NH) protecting group (PGz) is then cleaved togive the corresponding alcohol or amine of formula (16), which canundergo cyclization and cleavage all at once to yield a compoundaccording to the invention of formula (17). The orthogonal protections(PG1, PG2, PG3 and PG4 when a fourth amino-acid is introduced) of thecompound of formula (17) see FIG. 4 b can be removed to yield a compoundof formula (18).

The above example of synthesis deals with the preparation of librariesof the “family 2” type for which positions 1, 2 and 3 are filled.Libraries of “families 1, 3 and 4” type as shown in FIG. 1 can beprepared exactly in the same way. In “family 1” type libraries, an extraamino-acid is incorporated at position 4. In “family 3” type libraries,positions 3 and 4 are empty and in “family 4” type libraries positions2, 3 and 4 are empty.

Thus, it is possible to develop chemical libraries of dozens, hundreds,and even many thousands of discrete chemical compounds, in an efficientand reliable manner. This being the case, it is possible to use theselibraries as chemical intermediates for the preparation ofpharmaceutical compounds or for the identification of suchpharmaceutical compounds or other useful species. Some of thesecompounds could also be used without further modification. Accordingly,the ability to prepare such complex libraries in a reliable andpredictable fashion is highly desired.

The term “pharmaceutical” as used herein means the ability of thecompounds to provide some therapeutic or physiological beneficialeffect. As used herein, the term includes any physiologically orpharmacologically activity that produces a localized or systemic effector effects in animals including warm blooded mammals such as humans.Pharmaceutically active agents may act on the peripheral nerves,adrenegic receptors, cholinergic receptors, the skeletal muscles, thecardiovascular system, smooth muscles, the blood circulatory system,synoptic sites, neuroeffector junctional sites, endocrine and hormonesystems, the immunological system, the reproductive system, the skeletalsystem, the autocoid system, the alimentary and excretory systems, thehistamine system and central nervous systems as well as other biologicalsystems. Thus, compounds derived from compositions of the presentinvention may be used as sedatives, psychic energizers, tranquilizers,anticonvulsant, muscle relaxants, anti-Parkinson agents, analgesics,anti-inflammatories, local anesthetics, muscle contractants, antibiotic,antiviral, antiretroviral, antimalarials, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasities, neoplastics andchemotherapy agents. These compounds could further be used to treatcardiovascular diseases, central nervous system diseases, cancermetabolic disorders, infections and dermatological diseases as well asother biological disorders and infections.

Among the potential uses of the compounds according the presentinvention are uses in scientific research as research reagents. Inaccordance with the present invention, it is now possible to preparepluralities of compounds to create libraries of compounds for research.Such libraries are known to be useful and are important in the discoveryof new drugs. In view of the chemical and conformational diversities ofsuch compounds e.g. the large number of functionalizable sites, a verylarge number of different compounds can be prepared. Moreover, suchcompounds can be prepared differentially, that is, in such a fashionthat a population of known species can be prepared reliably, ensuringthat all potential members of a family of chemical species are in factsynthesized.

In view of the foregoing, persons of ordinary skill in the art will knowhow to synthesize such libraries, comprising chemical compositionswithin the scope and spirit of this invention and to assay the librariesagainst etiological agents or in tests or assays, in order to identifycompounds having antibacterial, antifungal, antiviral, antineoplastic orother desired pharmaceutical, biological, or chemical activity.

SPECIFIC EXAMPLES

Preparation of macrocyclic compounds according to the invention by theprocess outlined above will be illustrated by the following non-limitingspecific examples:

Example 1 Library of 135 Members Solution Phase See FIG. 2 a

This library (family 2 type) consists of a linear sequence of threenatural L-α-amino-acids linked together by an aliphatic chain with 4 or5 carbons in a head to tail manner. The first amino acids (AA₃) areglycine, leucine and methionine, the second ones (AA₂) are glycine,histidine (Doc), leucine, proline and valine, and the third ones (AA₃)are gylcine, methionine and phenylalanine.

The three third amino acids (Boc-AA3) were converted to their thioestersby coupling with methyl 3-mercapto-propionate. The formed compounds werecoupled with the second five amino acids to produce 15 dipeptides whichwere then converted to 135 linear tripeptides by coupling with nineN-alkylated N-betsyl amino-acids. The end-to-end cyclization of thelinear tripeptide thioesters was achieved by silver cation-assistedlactamization.

Amino Acid, Thioesters (Boc-AA₃-S(CH₂)₂CO₂Me):

Chlorotrimethylsilane (276 mL, 2.18 mol) was added slowly to a solutionof 3-mercaptopropionic acid (70 g, 0.66 mol) in methanol (267 mL, 6.6mol) at −10° C. The reaction mixture was then stirred for 1 h at 0° C.and for 1 h at room temperature. The mixture was neutralized withsaturated aqueous sodium bicarbonate to pH 8 and extracted withdichloromethane (3×300 mL). The combined organic phase was washed withsaturated aqueous sodium bicarbonate (2×200 mL), brine (2×200 mL), driedover magnesium sulfate, filtered and evaporated under reduced pressure.The residue was then distilled (70° C./20 mmHg) to give methyl3-mercaptopropionate as a colorless oil (61.2 g) in the yield of 77%.

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (42 mmol)was added to a solution of N-Boc amino-acid (33 mmol), methyl3-mercaptopropionate (30 mmol) and 4-dimethylaminopyridine (3 mmol) indichloromethane (80 mL) at 0° C. The resulting mixture was stirred for 1hour at 0° C. and 30 min at room temperature. The reaction mixture wasdiluted with ethyl acetate (200 mL), washed with 1N hydrochloric acid(2×50 mL), saturated aqueous sodium bicarbonate (2×50 mL), brine (2×50mL), dried over magnesium sulfate, and evaporated to give the desiredthioester (Boc-AA₃-S(CH₂)₂CO₂Me) in yields ranging from 95 to 100%.

Dipeptides (Boc-AA₃-S(CH₂)₂CO₂Me):

To a solution of the N-Boc amino acid thioester (Boc-AA₃-S(CH₂)₂CO₂Me)(5 mmol) in dichloromethane (2 mL), triethylsilane (10 mmol) was added,followed by trifluoro-acetic acid (3 mL). The reaction mixture wasstirred for 1 h at room temperature, then diluted with toluene (2×10 mL)and the solvent was evaporated to give the TFA salt ofH-AA₃-S(CH₂)₂CO₂Me.

To a solution of N-Boc amino acid (Boc-AA₂-OH) (5 mmol) and1-hydroxybenzotriazole (5 mmol) in tetrahydrofuran (5 mL) anddichloromethane (5 mL) at 0° C.,1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (7 mmol)was added. The resulting mixture was stirred for 5 min at 0° C. and for20 min at room temperature, then cooled down to 0° C.

A solution of the TFA salt of H-AA₃-S(CH₂)₂CO₂Me (5 mmol) indichloromethane (5 mL) was added to the above reaction mixture at 0° C.,followed by diisopropylethylamine (7.5 mmol). The resulting reactionmixture was then stirred at the same temperature for 10 min. Theice-water bath was removed and the reaction was stirred for 2 to 4 h atroom temperature. Finally, the reaction mixture was diluted with ethylacetate (80 mL) and washed with 1N hydrochloric acid (2×15 mL),saturated aqueous sodium bicarbonate (2×15 mL), brine (2×20 mL), driedover magnesium sulfate, and evaporated to give the dipeptide(Boc-AA₂-AA₃-S(CH₂)₂CO₂Me) in yields ranging from of 80 to 100%.

Alkylated Tripeptides (N-Bts-(N-alkylated)-AA₁-AA₂-AA₃-S(CH₂)₂CO₂Me):

To a solution of N-Boc dipeptide (0.5 mmol) in dichloro-methane (1 mL),triethylsilane (2 mmol) was added, followed by trifluoroacetic acid (1mL). The reaction mixture was stirred for 1 h at room temperature andthen diluted with toluene (2×5 mL) and evaporated to give the dipeptideTFA salt.

To a solution of alkylated N-Betsyl amino acid (0.5 mmol) and1-hydroxybenzotriazole (0.5 mmol) in tetrahydrofuran (2 mL) anddichloromethane (2 mL) at 0° C.,1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride (0.7 mmol)was added. The resulting mixture was stirred for 5 min at 0° C. then for30 min at room temperature, and cooled down to 0° C.

A solution of the dipeptide TFA salt (0.5 mmol) in dichloromethane (2mL) was added to the above reaction mixture at 0° C., followed bydiisopropylethylamine (0.8 mmol). The reaction mixture was then stirredfor 10 min at the same temperature. The ice-water bath was removed andthe reaction was stirred for 2 to 4 h at room temperature. The reactionmixture was diluted with ethyl acetate (50 mL) and washed with 1Nhydrochloric acid (2×10 mL), saturated aqueous sodium bicarbonate (2×10mL), brine (2×10 mL), dried over magnesium sulfate, and evaporated togive the alkylated tripeptides(Bts-(N-alkylated)-AA₁-AA₂-AA₃-S(CH₂)₂CO₂Me) with yields ranging from 11to 100%.

Preparation of Cyclotripeptides:

To a solution of the alkylated tripeptide(Bts-(N-alkylated)-AA₁-AA₂-AA₃-S(CH₂)₂CO₂Me) (0.5 mmol) indichloro-methane (1 mL), triethylsilane (2 mmol) was added, followed bytrifluoroacetic acid (1 mL). The reaction mixture was stirred for 1 h atroom temperature and then diluted with toluene (2×5 mL) and the solventwas evaporated to give the TFA salt of alkylated tripeptide.

To a solution of the TFA salt of alkylated tripeptide (0.5 mmol) inethyl acetate (250 mL), diisopropylethylamine (1.0 mmol) and silvertrifluoroacetate (1.5 mmol) were added. The reaction mixture was stirredfor 1 to 3 h at room temperature (Thin Layer Chromatography monitoringof the reaction). Brine (50 mL) and 1.0 M aqueous sodium thiosulfate (30mL) was added and stirred for 60 min. The organic phase was washed withsaturated EDTA aqueous solution (2×50 mL), 1N hydrochloric acid (50 mL),brine (2×50 mL), dried over magnesium sulfate and evaporated to give thecrude product. The crude can be purified by flash column chromatographyif necessary.

TABLE 1 Results for the cyclic peptides with the E-alkene linker. PurityPurity Quantity Yield Level Quantity Yield Level Name MW (mg) (%) (%)Name MW (mg) (%) (%) c-B-GGG-1 451 25 14 100 c-B-MLG-1 581 200 93 91c-B-GGM-1 525 170 77 94 c-B-MLM-1 655 162 61 99 c-B-GGF-1 541 60 44 96c-B-MLF-1 671 150 89 97 c-B-LGG-1 507 180 87 92 c-B-GPG-1 491 26 21 85c-B-LGM-1 581 95 40 95 c-B-GPM-1 565 121 nd 87 c-B-LGF-1 597 93 83 93c-B-GPF-1 581 80 62 89 c-B-MGG-1 525 80 35 96 c-B-LPG-1 547 73 63 97c-B-MGM-1 599 66 28 94 c-B-LPM-1 621 28 31 55 c-B-MGM-1 599 31 10 77c-B-LPF-1 637 25 9 85 c-B-MGF-1 615 82 27 97 c-B-LPF-1 637 135 64 92c-B-MGF-1 615 25 6 69 c-B-MPG-1 565 102 58 91 c-B-GHG-1 673 107 53 91c-B-MPM-1 639 10 98 99 c-B-GHM-1 747 97 59 98 c-B-MPF-1 655 140 61 76c-B-GHF-1 763 177 68 91 c-B-GVG-1 493 100 56 100 c-B-LHG-1 729 82 61 85c-B-GVM-1 567 13 9 59 c-B-LHM-1 803 133 65 80 c-B-GVF-1 583 142 75 80c-B-LHF-1 819 162 74 97 c-B-LVG-1 549 142 77 96 c-B-MHG-1 747 177 63 97c-B-LVM-1 623 78 95 90 c-B-MHM-1 821 160 44 90 c-B-LVF-1 639 290 85 89c-B-MHF-1 837 201 51 91 c-B-MVG-1 567 303 95 90 c-B-GLG-1 507 229 90 97c-B-MVM-1 641 26 66 96 c-B-GLM-1 581 146 68 99 c-B-MVF-1 657 222 64 85c-B-GLF-1 597 130 81 80 c-B-LLM-1 637 123 69 95 c-B-LLG-1 563 152 88 92c-B-LLF-1 653 221 97 96

TABLE 2 Results for the cyclic peptides with the Z-alkene linker.Quantity Yield Purity Quantity Yield Purity Name MW (mg) (%) (%) Name MW(mg) (%) (%) c-B-GGG-2 437 2 1 89 c-B-MLG-2 567 308 55 56 c-B-GGM-2 511250 77 86 c-B-MLM-2 641 38 5 28 c-B-GGF-2 527 46 46 96 c-B-MLF-2 657 20632 44 c-B-LGG-2 493 142 76 99 c-B-GPG-2 477 31 29 92 c-B-LGM-2 567 11223 55 c-B-GPM-2 551 76 nd 82 c-B-LGF-2 583 64 35 67 c-B-GPF-2 567 55 4292 c-B-MGG-2 511 56 49 96 c-B-LPG-2 533 0 nd c-B-MGM-2 585 28 10 87c-B-LPM-2 607 34 54 49 c-B-MGM-2 585 40 14 77 c-B-LPF-2 623 19 8 96c-B-MGF-2 601 75 20 94 c-B-MPG-2 551 46 22 78 c-B-MGF-2 601 32 9 88c-B-MPM-2 625 5 1 59 c-B-GHG-2 659 80 49 93 c-B-MPF-2 641 44 11 49c-B-GHM-2 733 52 22 63 c-B-GVG-2 479 61 23 85 c-B-GHF-2 749 159 39 66c-B-GVM-2 553 59 80 93 c-B-LHG-2 715 72 47 94 c-B-GVF-2 569 191 86 76c-B-LHM-2 789 111 31 51 c-B-LVG-2 535 92 36 89 c-B-LHF-2 805 169 46 67c-B-LVM-2 609 60 57 93 c-B-MHG-2 733 30 9 98 c-B-LVF-2 625 152 36 87c-B-MHM-2 807 90 6 42 c-B-MVG-2 553 112 30 82 c-B-MHF-2 823 159 30 69c-B-MVM-2 627 6 7 48 c-B-GLG-2 493 166 97 100 c-B-MVF-2 643 0 ndc-B-GLM-2 567 143 85 97 c-B-LLM-2 623 85 26 61 c-B-GLF-2 583 90 32 57c-B-LLF-2 639 193 43 77 c-B-LLG-2 549 110 48 94

TABLE 3 Results for the cyclic peptides with the alkyne linker. QuantityYield Purity Quantity Yield Purity Name MW (mg) (%) (%) Name MW (mg) (%)(%) c-B-GGG-3 435 11 6 96 c-B-LLG-3 547 124 46 83 c-B-GGM-3 509 258 5885 c-B-LLG-3 547 61 11 41 c-B-GGF-3 525 15 9 93 c-B-LLM-3 621 148 62 81c-B-LGG-3 491 150 69 91 c-B-LLF-3 637 196 55 82 c-B-LGM-3 565 60 22 98c-B-MLG-3 565 167 24 40 c-B-LGM-3 565 86 27 84 c-B-MLM-3 639 27 9 76c-B-LGF-3 581 36 34 91 c-B-MLF-3 655 100 22 65 c-B-LGF-3 581 10 8 91c-B-GPG-3 475 37 46 99 c-B-LGF-3 581 22 18 83 c-B-GPM-3 549 15 16 50c-B-MGG-3 509 60 38 89 c-B-GPF-3 565 45 83 99 c-B-MGM-3 583 32 12 96c-B-LPG-3 531 0 1 58 c-B-MGM-3 583 54 17 83 c-B-LPM-3 605 54 29 38c-B-MGF-3 599 96 31 98 c-B-LPF-3 621 26 10 91 c-B-MGF-3 599 55 13 73c-B-MPG-3 549 23 15 98 c-B-GHG-3 657 104 54 94 c-B-MPM-3 623 10 41 41c-B-GHM-3 731 52 26 61 c-B-MPF-3 639 5 nd c-B-GHF-3 747 80 24 81c-B-GVG-3 477 39 19 79 c-B-LHG-3 713 119 70 85 c-B-GVM-3 551 33 34 66c-B-LHM-3 787 173 66 73 c-B-GVF-3 567 36 6 28 c-B-LHF-3 803 224 64 75c-B-LVG-3 533 158 64 82 c-B-MHG-3 731 107 29 93 c-B-LVM-3 607 75 64 83c-B-MHM-3 805 97 13 78 c-B-LVF-3 623 356 64 79 c-B-MHF-3 821 163 38 83c-B-MVG-3 551 285 63 62 c-B-GLG-3 491 63 38 100 c-B-MVM-3 625 8 9 50c-B-GLM-3 565 53 15 51 c-B-MVF-3 641 123 31 77 c-B-GLF-3 581 20 7 44

Spectral Data for Betsylated c-Met-Leu-Phe-Linker Compounds:

Trans-Linker (c-B-MLF-1):

¹H NMR (CDCl₃, 300 MHz): 8.32 (1H, br), 8.19 (1H, dd, J=8.0 and 1.7 Hz),8.01 (1H, dd, J=8.5 and 1.7 Hz), 7.70-7.59 (2H, m), 7.34-7.22 (5H, m),6.91 (1H, br), 6.66 (1H, br), 5.70-5.60 (1H, m), 5.21-5.16 (1H, m),4.64-4.59 (2H, m), 3.92-3.85 (3H, m), 3.35-3.20 (2H, m), 3.30 (1H, dd,J=14.0 and 4.7 Hz), 3.16 (1H, dd, J=13.9 and 9.3 Hz), 2.47-2.32 (3H, m),2.09-2.04 (3H, m), 1.97 (3H, s), 1.73-1.44 (3H, m), 0.86 (3H, d, J=6.3Hz), 0.81 (3H, d, J=6.3 Hz).

LC-MS: m/e: 671 (M⁺)

Cis-Linker (c-B-MLF-2):

¹H NMR (CDCl₃, 300 MHz): 8.65 (1H, J=5.9 Hz), 8.05 (1H, dd, J=8.9 and1.6 Hz), 8.00 (1H, d, J=8.1 Hz), 7.66-7.57 (2H, m), 7.33-7.22 (3H, m),7.16 (2H, d, J=7.1 Hz), 6.71-6.68 (2H, m), 5.72-5.56 (2H, m), 5.01 (1H,t, J=7.3 Hz), 4.54 (1H, dt, J=8.7 and 4.9 Hz), 4.08-4.00 (2H, m), 3.94(1H, dd, J=16.4 and 7.3 Hz), 3.67-3.56 (2H, m), 3.23 (1H, dd, J=14.1 and4.8 Hz), 3.00 (1H, dd, J=14.1 and 9.0 Hz), 2.67-2.47 (2H, m), 2.36-2.11(1H, m), 2.09 (3H, s), 2.07-1.99 (1H, m), 1.92-1.82 (1H, m), 1.61-1.52(1H, m), 1.49-1.41 (1H, m), 0.93 (3H, d, J=6.5 Hz), 0.87 (3H, d, J=6.3Hz).

LC-MS: m/e: 657 (M⁺).

Acetylene-Linker (c-B-MLF-3):

¹H NMR (CDCl₃, 300 MHz): 8.32 (2H, d, J=8.1 Hz), 8.01 (1H, d, J=8.0 Hz),7.72-7.60 (2H, m), 7.49 (1H, br), 7.31-7.20 (5H, m), 6.80 (1H, br),4.80-4.76 (1H, m), 4.42-4.36 (2H, m), 4.13-3.95 (3H, m), 3.45-3.40 (1H,m), 3.29 (1H, dd, J=14.2 and 5.6 Hz), 3.14 (1H, dd, J=14.1 and 10.1 Hz),2.54-2.47 (3H, m), 2.12-2.08 (1H, m), 1.97 (3H, s), 1.74 (2H, t, J=7.6Hz), 1.42-1.33 (1H, m), 0.82 (3H, d, J=6.6 Hz), 0.78 (3H, d, J=6.6 Hz).

LC-MS: m/e: 655 (M⁺).

Example 2 Removal of Betsyl Group of Cyclotripeptides

1) In Solution:

Potassium trimethylsilanolate (0.2 mmol) was added to a solution of2-naphthalenethiol (0.2 mmol) in a deoxygenated mixture of THF and EtOH(1:1, 1 mL) at room temperature and the resulting mixture was stirredfor 20 min. N-Bts-cyclotripeptide (0.1 mmol) was then added. Theresulting mixture was stirred for 1 h and evaporated to dryness. Theresidue was purified by column to give the deprotected product withyields ranging from 77 to 86%.

2) On Solid Support:

Polystyrene-thiophenol Resin (0.2 mmol) was added to a solution ofpotassium trimethylsilanolate (0.2 mmol) in a deoxygenated mixture ofTHF and EtOH (1:1, 1 mL) at room temperature and the resulting mixturestirred for 20 min. The solution was removed by filtration. The resinwas washed with a deoxygenated mixture of THF and EtOH (1:1, 3×2 mL) andadded to a solution of N-Bts-cyclotripeptide (0.1 mmol) in adeoxygenated mixture of THF and EtOH (1:1, 1 mL). The resulting mixturewas stirred for 1 h. The resin was removed by filtration and washed withTHF and EtOH (1:1 mixture, 2×2 mL). The filtrate was concentrated togive the crude product in quantitive yield.

Data for c-Met-Leu-Phe-Linker Compounds:

Trans-Linker (c-MLF-1):

¹H NMR (CDCl₃, 300 MHz): 7.77 (1H, d, J=7.7 Hz), 7.30-7.14 (5H, m), 6.65(1H, d, J=7.4 Hz), 6.53 (1H, br), 5.56-5.64 (1H, m), 5.32-5.22 (1H, m),4.03-3.95 (2H, m), 3.79-3.68 (1H, m), 3.57-3.35 (3H, m), 3.12-3.02 (2H,m), 2.96-2.86 (1H, m), 2.61-2.50 (2H, m), 2.24-1.98 (3H, m), 2.10 (3H,s), 1.78 (2H, m), 1.55-1.39 (2H, m), 0.86 (3H, d, J=6.6 Hz), 0.83 (3H,d, J=6.8 Hz).

LC-MS: m/e: 474 (M⁺)

Cis-Linker (c-MLF-2):

¹H NMR (CDCl₃ & CD₃OD, 300 MHz): 7.27-7.14 (5H, m), 5.80-5.71 (1H, m),5.68-5.53 (1H, m), 4.21-4.09 (2H, m), 3.88 (1H, dd, J=14.0 and 6.0 Hz),3.64 (1H, dd, J=14.0 and 7.3 Hz), 3.34-3.15 (5H, m), 2.52 (2H, t, J=7.3Hz), 2.07 (3H, s), 2.00-1.86 (2H, m), 1.53-1.15 (3H, m), 0.80 (6H, d,J=6.6 Hz).

LC-MS: m/e: 460 (M⁺).

Acetylene-Linker (c-MLF-3):

¹H NMR (CDCl₃, 300 MHz): 7.92 (1H, d, J=8.8 Hz), 7.34 (1H, d, J=9.2 Hz),7.31-7.17 (5H, m), 7.02 (1H, t, J=5.9 Hz), 4.21 (1H, q, J=8.0 Hz),4.10-3.99 (2H, m), 3.79 (1H, dm, J=15.5 Hz), 3.67 (1H, dm, J=16.0 Hz),3.49-3.35 (3H, m), 3.25 (1H, dd, J=8.8 and 4.3 Hz), 2.60-2.54 (2H, m),2.15-1.99 (1H, m), 2.11 (3H, s), 1.80-1.68 (2H, m), 1.57-1.46 (2H, m),0.86 (6H, d, J=6.3 Hz).

LC-MS: m/e: 458 (M⁺).

Example 3 Preparation of N-Formyl-Cyclotripeptides

The free amine macrocycle (see example 2) was added to a mixture offormic acid (0.2 mL) and acetic anhydride (0.1 mL) and the reactionmixture was stirred for 15 min at room temperature, then for 5 min at55° C. and finally for 2 h at room temperature. The reaction mixture wasevaporated to dryness and purified by column chromatography to give thedesired compounds in yields ranging from 81 to 100%.

Data for Formylated c-Met-Leu-Phe-Linker Compounds:

Trans-Linker (c-f-MLF-1):

¹H NMR (CDCl₃, 300 MHz): 8.72 (1H, s), 7.92 (1H, s), 7.38-7.22 (5H, m),6.78 (1H, br), 5.83 (1H, d, J=10.0 Hz), 5.68-5.58 (1H, m), 4.96-4.89(1H, m), 4.02-3.68 (5H, m), 3.34 (1H, dd, J=14.0 and 10.0 Hz), 2.78 (1H,dd, J=13.5 and 5.2 Hz), 2.67-2.51 (3H, m), 2.42-2.31 (1H, m), 2.18-2.04(1H, m), 2.06 (3H, s), 1.99-1.53 (6H, m), 1.03 (3H, d, J=6.0 Hz), 0.94(3H, d, J=6.0 Hz).

LC-MS: m/e: 502 (M⁺) (84.2%).

Cis-Linker (c-f-MLF-2):

¹H NMR (CDCl₃, 300 MHz): 8.04 (1H, s), 7.29-7.18 (5H, m), 6.82-6.74 (1H,m), 6.39 (1H, d, J=9.5 Hz), 6.16 (1H, dd, J=18.0 and 7.5 Hz), 6.94-6.85(1H, m), 4.80-4.72 (1H, m), 4.24-3.68 (5H, m), 3.50-3.43 (1H, m),2.87-2.79 (1H, m), 2.61-2.45 (3H, m), 2.14-2.01 (1H, m), 2.11 (3H, s),1.74-1.62 (1H, m), 1.43-1.25 (3H, m), 0.93-0.78 (6H, m).

LC-MS: m/e: 488 (M⁺).

Acetylene-Linker (c-f-MLF-3):

¹H NMR (CDCl₃, 300 MHz): 8.10 (1H, s), 7.83 (1H, br), 7.31-7.16 (5H, m),6.97 (1H, br), 6.45 (1H, br), 4.35-4.01 (5H, m), 3.87-3.71 (2H, m), 3.38(1H, dd, J=14.5 and 5.0 Hz), 3.24-3.16 (1H, m), 2.59-2.23 (3H, m), 2.08(3H, s), 1.66-1.25 (4H, m), 0.84 (6H, t, J=6.0 Hz).

LC-MS: m/e: 486 (M⁺).

Example 4 Solid Phase Synthesis on the Kaiser Resin

Anchoring Boc-Amino Acid to the Resin:

To the Kaiser resin (2.0 g, 0.95 mmol/g) was added a 0.2M solution ofN-Boc amino acid and 4-dimethylaminopyridine (0.25 eq). After shakingfor 5 min, diisopropylcarbodiimide (1.5 eq) was added and the reactionmixture was agitated for 16 h. The resin was washed with dichloromethane(3×30 mL), tetrahydrofuran (1×30 mL), methanol (1×30 mL),dichloromethane (1×30 mL), methanol (1×20 mL), tetrahydrofuran (1×30mL), methanol (1×20 mL), dichloromethane (2×30 mL) and dried by nitrogenflow. The unreacted hydroxy group on the resin was then capped byreacting with acetic anhydride (5 mL) and diisopropyl-ethylamine (1 mL)in dichloromethane (20 mL) at room temperature for 2 h. The resin waswashed and dried by the same procedure mentioned above. The substitutionlevel was 0.2-0.3 mmol/g.

Formation of Dipeptides (Performed on the Quest 210™):

25% TFA in dichloromethane (20 mL) was added to the above resin (0.4mmol, 2.0 g, 0.2 mmol/g) and agitated for 30 min. The resin was thenwashed with dichloromethane (3×30 mL), methanol (1×20 mL),dichloromethane (1×30 mL), methanol (1×20 mL), dichloromethane (1×30mL), methanol (1×20 mL), dichloromethane (2×30 mL) and dried by nitrogenflow.

A 0.2M solution of hydroxybenzotriazole and diisopropyl-carbodiimide in60% dichloromethane/tetrahydrofuran was added to the N-Boc amino acid,followed by diisopropyl-ethylamine (1.5 eq). The resulting mixture wasstirred for 30 min at room temperature, and then transferred to theresin (200 mg on Quest 210™) and agitated at room temperature for 30min. Diisopropylethylamine (2.0 mmol) was then added and agitated untilKaiser test of an aliquot of the resin was negative (2 to 4 h). Theresin was washed with dichloromethane (3×4 mL), tetrahydrofuran (1×4mL), methanol (1×4 mL), dichloromethane (1×4 mL), methanol (1×4 mL),tetrahydrofuran (1×4 mL), methanol (1×4 mL), dichloromethane (2×4 mL)and dried by nitrogen flow.

Formation of Tripeptides:

25% TFA in dichloromethane (4 mL) was added to the Boc-protecteddipeptide resin (0.04 mmol, 200 mg g, 0.2 mmol/g) and agitated for 30min. The resin was then washed with dichloromethane (3×4 mL), methanol(1×4 mL), dichloro-methane (1×4 mL), methanol (1×4 mL), dichloromethane(1×4 mL), methanol (1×4 mL), dichloromethane (2×4 mL) and dried bynitrogen flow.

A 0.2M solution of hydroxybenzotriazole and diisopropyl-carbodiimide in60% dichloromethane/tetrahydrofuran was added to the N-Bts amino acid,followed by diisopropyl-ethylamine (1.5 eq). The resulting mixture wasstirred for 30 min at room temperature, and then transferred to theresin (200 mg on Quest 210™) and agitated at room temperature for 30min. The resin was washed with dichloromethane (3×4 mL), tetrahydrofuran(1×4 mL), methanol (1×4 mL), dichloromethane (1×4 mL), methanol (1×4mL), tetrahydrofuran (1×4 mL), methanol (1×4 mL), dichloromethane (2×4mL) and dried by nitrogen flow.

Mitsunobu Reaction:

A 0.2M solution of 5-(tert-butoxycarbonylamino)-trans-2-penten-1-ol orN-tert-butoxycarbonyl-2-(2-hydroxyethoxy)-cinnamyl amine, andtriphenylphosphine was added to the tripeptide resin (0.036 mmol, 180mg, 0.2 mmol/g on the Quest 210™) in anhydrous tetrahydrofuran, followedby diethyl azodicarboxylated (1.5 eq). The mixture was agitated for 2 hand the resin was washed and dried by the same procedure mentionedabove.

Cyclization of Alkylated Tripeptides:

The N-alkylated linear tripeptide (0.016 mmol, 80 mg) was treated with25% TFA in dichloromethane (4 mL) for 30 min and washed withdichloromethane, methanol and dried by nitrogen flow.

Toluene (2 mL), acetic anhydride (1 mL) and diisopropyl-ethylamine (1mL) were added to the above resin and agitated for 2 h. The resin wasremoved by filtration and washed with dichloromethane (3×4 mL). Analiquot of the filtrate was analysized by LC-MS. The filtrate wasconcentrated and then diluted with ethyl acetate (15 mL). The solutionwas washed with 1N hydrochloric acid (2 mL), saturated sodiumbicarbonate (2 mL), brine (3 mL), dried and evaporated to give the crudeproduct.

TABLE 4 Library of Macrocyclic Tripeptides Synthesized on the KaiserResin Using the Quest 210 ™: ISOLATED SAMPLE ID QUANTITY (MG) PURITYc-B-MPG-1 5.9 (42%) Low c-B-MVG-1 4.4 (48%) Medium c-B-MGM-1 4.6 (32%)Low c-B-LPM-1 7.5 (50%) Low c-B-LLM-1 7.1 (46%) Good c-B-GVM-1 3.1 (22%)Low c-B-GLF-1 7.8 (81%) Good c-B-MVG-4* 2.4 (23%) Good c-B-MGM-4* 5.7(34%) Low c-B-LPM-4* 3.3 (19%) Low c-B-LMM-4* 5.0 (29%) Mediumc-B-GVM-4* 4.0 (25%) Low c-B-GLF-4* 2.9 (26%) Low *Nature of linker 4 isshown in Scheme 2.

Example 5 Synthesis of Betsylated Macrocyclic Tripeptides on Thio-EsterResin in IRORI MACROKANS™

Macrocyclic tripeptides were synthesized in MACROKANS™ (160 mg ofaminomethyl resin) following the same procedure as that given for thesynthesis on solid support (Kaiser Resin, see example 4):

TABLE 5 Library of Macrocyclic Tripeptides Synthesized SAMPLE IDQUANTITY (MG) PURITY c-B-GHG-1 7.6 (6%) Good c-B-GLF-1 16.3 (14%)Excellent c-B-GVM-1   20 (18%) Good c-B-LHF-1   43 (27%) Excellentc-B-LPM-1 26.7 (22%) Excellent c-B-LLM-1 20.6 (17%) Excellent c-B-MVG-110.5 (10%) Good c-B-MPG-1 6.4 (6%) Low c-B-GHG-4 12.4 (11%) Excellentc-B-GLF-4 12.4 (9%)  Excellent c-B-GVM-4 9.3 (7%) Good c-B-LHF-4 21.8(16%) Excellent c-B-LPM-4 29.1 (21%) Excellent c-B-LLM-4 25.8 (18%)Excellent c-B-MVG-4 37.5 (30%) Excellent c-B-MPG-4 30.3 (24%) Excellent

Example 6 Synthesis of Mesylated Macrocyclic Tripeptides on ThioesterResin in IRORI MINIKANS™

Macrocyclic tripeptides were synthesized in MINIKANS™ (60 mg ofaminomethyl resin) following the same procedure as that given for thesynthesison solid support (Kaiser Resin, see example 4):

TABLE 6 Library of Macrocyclic Tripeptides Synthesized SAMPLE IDQUANTITY (MG) PURITY c-Ms-GLG-4 3 (9%)  Low c-Ms-GLG-1 3 (12%) Lowc-Ms-E(OMe)LG-4 1.9 (5%)   Good c-Ms-E(Ome)LG-1 4 (13%) Good c-Ms-LPM-40.1 Good c-Ms-LPM-1 0.5 Low c-Ms-YLG-4 2 (5%)  Good c-Ms-YLG-1 6 (18%)Good c-Ms-GLF-4 6 (16%) Good c-Ms-GLF-1 5 (15%) Good c-Ms-LLG-4 1 (3%) Low c-Ms-LLG-1 — Low c-Ms-LLM-4 — Low c-Ms-SLG-4 — Good c-Ms-E(COOH)LG-413.3* Good c-Ms-E(COOH)LG-1   21* Good *Unpurified

Specific Examples of Building Units

Tether Building Blocks (Type D, See FIG. 2 b)

The tether building blocks have the following formula: HO—Y-L-Z-PGz.

The three following tether building blocks4-(tert-butoxycarbonylamino)-cis-2-buten-1-ol (Y═CH₂, L=[Z]alkene,Z═CH₂) and 4-(tert-butoxycarbonylamino)-2-butyn-1-ol (Y═CH₂, L=alkyne,Z═CH₂) and 5-(tert-butoxycarbonylamino)-trans-2-penten-1-ol (Y═CH₂,L=CH₂-[E]alkene, Z═CH₂) were synthesized from commercial availablecis-2-butene-1,4-diol, 2-butyne-1,4-diol and 3-amino-1-propanolrespectively.

Preparation of 4-(Tert-Butoxycarbonylamino)-2-Butyn-1-ol:

Dowex 50WX8-100 ion-exchange resin (53 g) was added to a suspension of2-butyne-1,4-diol (153.9 g, 1.77 mol) and dihydropyran (250 mL, 2.66mol) in dichloromethane (800 mL) at room temperature. The resultingmixture was stirred for 60 min and quenched with triethylamine (10 mL).The resin was removed by filtration. The filtrate was washed with asaturated aqueous solution of sodium bicarbonate (100 mL), brine (3×300mL), dried over magnesium sulfate, filtered and evaporated under reducedpressure to give the desired monoprotected 2-butyne-1,4-diol with ayield of 50%.

To a solution of monoprotected 2-butyne-1,4-diol (20.4 g, 0.12 mol) andtriphenylphosphine (40.9 g, 0.16 mol) in tetrahydrofuran (50 mL),hydrazoic acid (113 mL, 1.6 M in toluene, 0.18 mol) was added at 0° C.Diisopropyl azodi-carboxylate (29.5 mL, 0.15 mol) was added dropwise tothe solution whose temperature was around 0° C. The reaction was stirredfor 30 min at 0° C. and for 30 min at room temperature.Triphenylphosphine (40.9 g, 0.16 mol) was added at 0° C. and thereaction was stirred overnight at room temperature. After addition ofwater (50 mL), the mixture was heated at 60° C. for 4 h, 1N hydrochloricacid (140 mL) was added. After stirring for 1 h, brine (140 mL) anddichloromethane (500 mL) were added. The aqueous phase was washed withdichloromethane (3×100 mL). Potassium carbonate (16.5 g, 0.12 mol) wasadded, followed by a solution of di-tert-butyl dicarbonate (26.2, 0.12mol) in tetrahydrofuran (100 mL). The reaction mixture was stirred for18 h, then extracted with dichloromethane (1×300 mL, 2×50 mL). Thecombined organic extract was washed with brine (50 mL), dried overmagnesium sulfate, filtered and evaporated under reduced pressure. Theresidue was purified by a dry-pack silica gel column to give the titledcompound in 50% yield.

Preparation of 4-(tert-butoxycarbonylamino)-cis-2-buten-1-ol

The title compound was synthesized from cis-2-butene-1,4-diol in anoverall yield of 30% according to the procedure used for4-tert-butoxycarbonylamino-2-butyn-1-ol.

Preparation of 5-(tert-butoxycarbonylamino)-trans-2-penten-1-ol

A solution of di-tert-butyl dicarbonate (382.8 g, 1.75 mol) indichloromethane (1.6 L) was added to 3-amino-1-propanol (263.6 g, 3.51mol) during 2 h. The reaction mixture was stirred for an additional 40min and water (1 L) was added. The organic phase was washed with water(3×500 mL),), dried over magnesium sulfate, filtered and evaporatedunder reduced pressure to give 3-(tert-butoxycarbonylamino)-1-propanolin 96% yield.

Dimethyl sulfoxide (321 mL, 4.5 mol) and triethylamine (941 mL, 6.75mol) were added to a solution of 3-tert-butoxycarbonylamino-1-propanol(262.6 g, 1.5 mol) in dichloromethane (1.2 L) at 0° C. Sulfur trioxidepyridine complex (286.4 g, 1.8 mol) was added in 6 portions. Thereaction mixture was then stirred for 30 min at 0° C. and for 30 min atroom temperature. The reaction was cooled down to 0° C., quenched with1N hydrochloric acid, washed with brine (2×500 mL), dried over magnesiumsulfate, filtered and evaporated under reduced pressure to give3-(tert-butoxycarbonylamino)propionaldehyde.

To a solution of the above crude aldehyde (1.5 mol) in acetonitrile (1.3L), trimethyl phosphonoacetate (409.7 g, 2.25 mol) and lithium hydroxide(53.9 g, 2.25 mol) were added and stirred overnight at room temperature.The reaction was quenched with water (50 mL).

The acetonitrile solvent was removed by evaporation under reducedpressure. The residue was then diluted with diethyl ether (800 mL),washed with 1N sodium hydroxide (3×300 mL), brine (2×500 mL), dried overmagnesium sulfate, filtered and evaporated under reduced pressure togive the desired trans-unsaturated methyl ester.

Diisobutylaluminium hydride (1.12 L, 1.0 M in dichloro-methane, 1.12mol) was added dropwise to a solution of this methyl ester (102.75 g,0.445 mol) in dichloromethane (250 mL) at 0° C. The resulting mixturewas stirred for an additional 1 h and then poured slowly into a 1Mtartaric acid aqueous solution (1.4 L), extracted with dichloro-methane(3×500 mL). The combined organic phase was dried over magnesium sulfate,filtered and evaporated under reduced pressure. The residue was purifiedby dry-pack silica gel column to give the titled compound with a yieldof 40% for the three steps.

Amino-Acid Building Block (Type A and B, See FIG. 2 a)

The N-Boc amino acids are commercial available exceptN^(∝)-Boc-N^(im)-Doc-histidine which was preparated by the methoddescribed below.

Preparation of N^(∝)-Boc-N^(im)-Doc-Histidine

A solution of 2,4-dimethyl-3-pentanol (83.2 g, 0.72 mol) andtriethylamine (125 mL, 0.90 mol) in toluene (300 mL) was added slowly(30 min) to a solution of phosgene (531 mL, 20% in toluene, 1.07 mol) intoluene (531 mL) at 0° C. The mixture was stirred for an additional 30min at the same temperature. Ice-cold water (500 mL) was added and theorganic phase was washed with ice-cold water (2×100 mL), dried overmagnesium sulfate, filtered and evaporated under reduced pressure. Theresidue was then distilled under reduced pressure (6 cm of Hg, 33-35°C.) to give Doc-Cl as a colorless oil (92 g, 72%).

A solution of Doc-Cl (57.9 g, 0.32 mol) in tetrahydrofuran (250 mL) wasadded slowly to a solution N^(∝)-Boc-histidine (69 g, 0.27 mol) andpotassium carbonate (41.1 g, 0.28 mol) in water (350 mL) at 0° C. Theresulting mixture (pH=10) was stirred for 2 h at the same temperatureand for 1 h at room temperature. Water (150 mL) and hexane (200 mL) wereadded to the reaction mixture. The separated aqueous phase was washedwith a mixture of diethyl ether and hexane (1:1; 3×200 mL), acidifiedwith 20% citric acid aqueous solution to pH 2-3, and then extracted withdichloromethane (3×200 mL). The combined dichloromethane solution waswashed with brine (200 mL), dried over magnesium sulfate, filtered andevaporated under reduced pressure to give the crude1\r-Boc-N^(im)-Doc-histidine in quantitive yield.

Building Blocks of Type C (See FIG. 2 b)

N-Betsyl protected amino acids (Bts-AA1) were synthesized by thereaction of amino acids with Betsyl chloride(benzothiazole-2-sulfonylchloride) which was obtained frommercaptobenzothiazole and chlorine.

Preparation of N-Betsyl Amino Acid (Bts-AA₁OH)

Chloride gas was bubbled into a solution of acetic acid (250 mL) inwater (500 mL) at 5° C. until an orange precipitate was formed in goodquantity. A solution of mercaptobenzothiazole (0.7 mol) in aqueousacetic acid (750 mL, 33% in water) was added in portions to the abovereaction mixture in a period of 3 hours. The reaction mixture wasstirred for 1 h, filtered at 0° C. then washed with cold water. Thesolid was dissolved in cold dichloromethane (500 mL) and washed withcold brine (2×100 mL), cold saturated sodium bicarbonate (100 mL), brine(100 mL), dried over magnesium sulfate, filtered and evaporated at 10°C. under reduced pressure. The solid was then washed with cold diethylether (100 mL), cold acetonitrile (100 mL), filtered and pumped to givebetsyl chloride (benzothiazole-2-sulfonylchloride).

To a solution of amino acid (0.11 mol) in 0.25 N aqueous sodiumhydroxide (0.08 mol) at room temperature (initial pH around 9.5), betsylchloride (0.1 mol) was added. The resulting mixture was stirredvigorously for 18 h. The pH of the reaction was adjusted between 9.5 to10.0 with 1.0 N aqueous sodium hydroxide during the reaction progress.The reaction mixture was washed with diethyl ether (3×50 mL). Theaqueous phase was then cooled to 0° C., acidified to pH 1.5-2.0 with 6 NHCl, and extracted with ethyl acetate (3×100 mL). The combined ethylacetate solution was dried over magnesium sulfate, filtered andevaporated under reduced pressure to give the desired compound in 74-85%yield.

Building Blocks of Type E (See FIG. 2 b)

These building blocks correspond to building blocks of type C which havebeen alkylated using Mitsunobu reaction conditions with the buildingblocks of type D. To be able to carry out this alkylation, the acidfunctional group of C must be protected. The protecting group used isfinally removed to get the desired compounds

Preparation of N-Alkylated N-Betsyl Amino Acid

Dihydrofuran (90 mmol) and pyridinium p-toluenesulfonate (1.5 mmol) wereadded to a suspension of N-Betsyl amino acid (30 mmol) indichloromethane (50 mL) at 0° C. The resulting mixture was stirred for15 min at 0° C. and for 60 min at room temperature. The reaction mixturewas diluted with diethyl ether (150 mL), washed with saturated aqueoussodium bicarbonate (20 mL), brine (20 mL), dried over magnesium sulfate,filtered and evaporated under reduced pressure to give thetetrahydrofuranyl ester of amino acid. A mixture of this ester (30mmol), an alcohol (type D building block) (i.e.4-(tert-butoxycarbonylamino)-cis-2-buten-1-ol, or4-(tert-butoxycarbonylamino)-2-butyn-1-ol, or5-(tert-butoxycarbonylamino)-trans-2-penten-1-ol)) (43.5 mmol) andtriphenylphosphine (49 mmol) were suspended in toluene (20 mL) andazeotropically distilled three times in vacuum. The residue wasdissolved in tetrahydrofuran (40 mL). Diisopropyl azodicarboxylate (43.5mmol) was added at 0° C. After stirring for 15 min at 0° C. and for 30min at room temperature, 1N hydrochloric acid (30 mL) and methanol (30mL) was added and stirred for an additional 60 min. After removal of theorganic solvents by evaporation, the aqueous phase was diluted withwater (30 mL), the pH of the medium was adjusted to 12 with potassiumcarbonate, washed with diethyl ether (3×60 mL), and then acidified to pH2-3 with 1N hydrochloric acid, extracted with dichloromethane (3×200mL). The combined dichloromethane solution was washed with brine (100mL), dried over magnesium sulfate, filtered and evaporated under reducedpressure to give the desired alkylated N-Betsyl amino acid in theoverall yield of 68-89%.

1-6. (canceled)
 7. A macrocyclic compound of formula I:

and pharmaceutically acceptable salts thereof, wherein A₁ is

where the nitrogen is bonded to T and the carbonyl is bonded to A₂; x is0, 1 or 2; R₁ is selected from the group consisting of:

wherein (P) indicates the point of attachment to CH; W₁ to W₁₆ are eachindependently hydrogen or a protecting group used for orthogonalprotection in peptide synthesis; and X is selected from the groupconsisting of:

A₂ is selected from the group consisting of prolinyl, 4-hydroxyprolinyl,4-tert-butoxyprolinyl; and

where the nitrogen is bonded to the carbonyl of A₁ and the carbonyl isbonded to A₃; wherein y is 0, 1 or 2; R₂ is selected from the groupconsisting of:

wherein (P) indicates the point of attachment to CH; and W₁ to W₁₆ areeach independently hydrogen or a protecting group used for orthogonalprotection in peptide synthesis; A₃ is selected from the groupconsisting of prolinyl, 4-hydroxyprolinyl, 4-tert-butoxyprolinyl; and

where the nitrogen is bonded to the carbonyl of A₂ and the carbonyl isbonded to T; wherein z is 0, 1 or 2; R₃ is selected from the groupconsisting of:

wherein (P) indicates the point of attachment to CH; and W₁ to W₁₆ areeach independently hydrogen or a protecting group used for orthogonalprotection in peptide synthesis; and (T) is selected from the groupconsisting of:

wherein (A₃) indicates the site of a covalent bond to A₃ of formula Iand (A₁) indicates the site of a covalent bond to A₁ of formula I. 8.The macrocyclic compound of claim 7, wherein x, y and z are eachindependently 0 or 1, and X is H.
 9. The macrocyclic compound of claim8, wherein R₁, R₂ and R₃ are each independently